Fueling the Future with Fungal Genomics Igor V. Grigoriev1*, Daniel Cullen2, David Hibbett3, Stephen B. Goodwin4, Thomas W Jeffries2, Christian P. Kubicek5, Cheryl Kuske6, Jon K. Magnuson7, Francis Martin8, Joey Spatafora9, Adrian Tsang10, Scott E. Baker1,7 1 US DOE Joint Genome Institute, Walnut Creek, California, USA 2 USDA, Forest Service, Forest Products Laboratory, Madison, WI, USA 3 Clark University, Worcester, MA, USA 4 USDA-Agricultural Research Service, Purdue University, West Lafayette, Indiana,USA 5 Institute of Chemical Engineering, TU Wien, Vienna, Austria; 6 Los Alamos National Laboratory, Los Alamos, NM, USA 7 Chemical and Biological Process Development Group, Pacific Northwest National Laboratory, WA, USA 8 INRA, Tree-Microbe Interactions Joint Unit, 54280 Champenoux, France 9 Oregon State University, OR, USA 10 Concordia University, Montreal, QC, Canada *To whom correspondence may be addressed. Igor V. Grigoriev, US DOE Joint Genome Institute, 2800 Mitchell Drive, Walnut Creek, CA 94598, USA. Telephone: 1.925.296.5860, Fax: 1.925.296.5752. Email: I. V. Grigoriev ([email protected]). 29 April 2011 ACKNOWLEDGMENTS: The work conducted by the U.S. Department of Energy Joint Genome Institute is supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. DISCLAIMER: This document was prepared as an account of work sponsored by the United States Government. While this document is believed to contain correct information, neither the United States Government nor any agency thereof, nor The Regents of the University of California, nor any of their employees, makes any warranty, express or implied, or assumes any legal responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any specific commercial product, process, or service by its trade name, trademark, manufacturer, or otherwise, does not necessarily constitute or imply its endorsement, recommendation, or favoring by the United States Government or any agency thereof, or The Regents of the University of California. The views and opinions of authors expressed herein do not necessarily state or reflect those of the United States Government or any agency thereof or The Regents of the University of California Fueling the Future with Fungal Genomics Igor V. Grigoriev1*, Daniel Cullen2, David Hibbett3, Stephen B. Goodwin4, Thomas W Jeffries2, Christian P. Kubicek5, Cheryl Kuske6, Jon K. Magnuson7, Francis Martin8, Joey Spatafora9, Adrian Tsang10, Scott E. Baker1,7 1 US DOE Joint Genome Institute, Walnut Creek, California, USA 2 USDA, Forest Service, Forest Products Laboratory, Madison, WI, USA 3 Clark University, Worcester, MA, USA 4 USDA-Agricultural Research Service, Purdue University, West Lafayette, Indiana,USA 5 Institute of Chemical Engineering, TU Wien, Vienna, Austria; 6 Los Alamos National Laboratory, Los Alamos, NM, USA 7 Chemical and Biological Process Development Group, Pacific Northwest National Laboratory, WA, USA 8 INRA, Tree-Microbe Interactions Joint Unit, 54280 Champenoux, France 9 Oregon State University, OR, USA 10 Concordia University, Montreal, QC, Canada * Please send correspondence to Igor V. Grigoriev, US DOE Joint Genome Institute, 2800 Mitchell Drive, Walnut Creek, CA 94598, USA, phone 1-925-296-5860, fax 1-925-296-5752, email [email protected] Fueling the Future with Fungal Genomics Fungi play important roles across the range of current and future biofuel production processes. From crop/feedstock health to plant biomass saccharification, enzyme production to bioprocesses for producing ethanol, higher alcohols or future hydrocarbon biofuels, fungi are involved. Research and development are underway to understand the underlying biological processes and improve them to make bioenergy production efficient on an industrial scale. Genomics is the foundation of the systems biology approach that is being used to accelerate the research and development efforts across the spectrum of topic areas that impact biofuels production. In this review, we discuss past, current and future advances made possible by genomic analyses of the fungi that impact plant/feedstock health, degradation of lignocellulosic biomass and fermentation of sugars to ethanol, hydrocarbon biofuels and renewable chemicals. Keywords: biofuels, genomics, fungi, Introduction Around the world, rapidly growing populations increase the demand for energy. Limited fossil resources and negative ecological impacts of petroleum exploration, extraction, transport, and consumption dictate the need to explore and develop alternative renewable sources of energy. One of the alternatives, cellulosic biofuels, is based on the combined efficiency of plants to produce and store carbon in the form of cellulose and other biopolymers such as hemicelluloses and lignin in their cell walls and of microbes to decompose these biopolymers and convert the resulting sugar building blocks to renewable fuels, fuel precursors and chemicals. Genomics offers the tools to explore the molecular mechanisms of these natural processes and to engineer industrial analogs. Several bioprocesses, which currently produce biofuels, biofuel precursors and renewable chemicals, are fungal based. The first major fungal genomics milestone was the publication of the whole genome sequence of the yeast Saccharomyces cerevisiae (Goffeau et al. 1996), an organism which has played an exceptional role in expanding our basic knowledge of eukaryotic cell physiology, genetics and biochemistry as well as brewing, baking and industrial ethanol production. The latter is one of the final steps in converting biomass to a biofuel – sugar fermentation to alcohol. Another industrially utilized and sequenced fungus, Trichoderma reesei (Martinez et al. 2008), produces large amounts of enzymes employed in breaking cellulose into simple sugars (glucose and xylose). A significant number of fungal genomes sequenced so far as well as many of those in progress are of medical importance (Cuomo and Birren 2010). Many fungi that are important to bioenergy and the environment still do not have sequenced genomes. To generate and understand the biology underlying fungal bioprocesses for producing enzymes, biofuels and chemicals we need to start with decoding the genomic blueprint of these organisms, identify parts lists - enzymes, pathways, and fungal hosts - and design industrial processes utilizing these parts (Baker et al. 2008). The U.S. Department of Energy (DOE) Joint Genome Institute (JGI) has launched a Fungal Genomics Program (FGP) aiming to scale up sequencing and analysis of fungal genomes to explore their diversity and applications for energy and environmental science. We started with several successful single-genome sequencing projects (e.g., Martinez et al. 2004, 2008, 2009; Jeffries et al. 2007; Martin et al 2008) and are moving towards larger-scale system-level genomics. Genomes and transcriptomes to be sequenced are selected from user community proposals to annual calls of the JGI Community Sequencing Program (e.g., Martin et al 2011). The sequenced and annotated genomes are deposited to MycoCosm (http://www.jgi.doe.gov/fungi), a web-based fungal genomics resource. This resource integrates genomics data and tools for comparative genomics and genome-centric analysis and promotes user community participation in data submission, annotation and analysis. The key project of FGP, the Genomic Encyclopedia of Fungi, focuses on three areas of research connected to bioenergy: (1) Plant Feedstock Health, encompassing symbiosis, plant pathogenicity, and biocontrol; (2) Biorefinery, analyzing lignocellulose degradation, sugar fermentation, and industrial organisms; and (3) Fungal Diversity. Sustainable production of feedstock plants such as switchgrass or poplar depends on plant interactions with fungal symbionts and pathogens. Converting biomass into biofuel requires a detailed inventory of enzymes and processes developed in filamentous fungi for saccharifying plant material and producing hydrocarbon biofuels and in yeasts that ferment sugars to ethanol, higher alcohols and hydrocarbon biofuels. Knowledge of the molecular biology of organisms serving as industrial hosts in biorefineries is critical. Developing robust industrial processes and their optimization requires organisms and enzymes that are tolerant to a range of temperatures, physical and chemical conditions, with lower maintenance and higher yield of desired product. These could come from a broader sampling of the phylogenetic and ecological diversity of fungi. Here we summarize current and planned genomics efforts in each chapter of the Genomic Encyclopedia of Fungi. 1. Feedstock Energy crops, such as Miscanthus, switchgrass, cottonwoods (hybrid poplars), hybrid willows and sugarcane are grown specifically for their ability to generate energy. In addition, corn and sorghum can also be grown for fuel, while the leftover by-products serve other purposes such as feed or food. Plant health maintenance is critical for sustainable growth of biofuel feedstocks and fungi, as symbionts, pathogens, or biocontrol agents, play a very important role. Symbionts such as mycorrhizae can increase productivity of bioenergy feedstock plants. Pathogens can have dramatic negative effects on yields, quality and production of bioenergy crops (Ullstrup 1972). Feedstock protection also can be achieved by biocontrol fungi,